different analysis methods used todetermination amount of histamine in fish product especially tuna fish which isthe most kind of fish consumed in the middle east because it’s the cheapesttype comparing to another types , these analysis methods used to determinehistamine are HPLC ,GC, TLC and CE. the comparison between these methoddone according to different parameters : sample pretreatment, derivatization required, column used , staitarinay phase used , mobilephase used , time analysis, lower detection limit , detector , linearity , andReagent used . Key word: histamine, HPLC,TLC, CE, GC. IntroductionHistamine is a chemical messengerthat mediates several cellular responses: inflammatory reactions, allergic reaction,gastric and acid secretion, limited neurotransmitter action in the brain.Histamine and receptors are presentessentially in all tissue types, there are especially high concentration ofhistamine receptors on cells found in the lungs , skin, blood vessels, an GItract .
histamine is stored in granules in mast cells throughout the body ,histamine can be released in response totissue injury resulting from cold , heat, toxins ( microbial organism ) , venom( snacks , insect ) , trauma and ingested histamine rich food . when histaminereleased it can bind to histamine receptors ( H1,H2,H3,H4) the most commonhistamine receptors (H1 and H2) , several effect are possible happenedaccording to tissue type and locationwhen histamine bind to (H1, H2 ) receptors .( see table one) . Organs located Type of histamine receptors Effects Peripheral sensory neurons H1 Itching, sometimes pain. Intestinal smooth muscle H1 Contraction, cramps, diarrhea. Secretary mucosa H1 Production mucus, rhinorrhea, coughs. Pulmonary smooth muscle H1 Decrease lung capacity and asthma.
Cardiovascular H1,H2 Hypotension, increase heart beat. Permatologic H1,H2 Leakage of fluid and protein into tissue. GI tract H2 Stimulate gastric acid secretion that lead to peptic ulcer.
Table 1: effects that present frombinding histamine to histamine receptors.Diamine oxidase enzyme ( DAO) located in the intestinal mucosa mainly becomeactive during the digestion of histamine rich food , during metabolism of histamine and if there is notenough DAO activity( the main cause of enzymatic dysfunction has a geneticorigin ) the imbalance between ingested histamine and the histamine releasedfrom the storage cells leads to histamine accumulation in plasma , that’slead to adverse effect on health ( histamineintolerance , histamine toxication ) , that leads to the following symptoms :diarrhea , headache , asthma , flushing , irregular heartbeat, hypotension ,and red welts on the skin , to reduce these problem avoid histamine rich foodthat’s shown in( table 2 ). Plant source Citrus food, papaya, strawberries, pineapple, nuts, peanuts, tomatoes, spinach, chocolate. Animal source Fish , shrimp , egg white others Additives, spices. Table 2: histamine rich food.
histamine produce in food bydecarboxylation of histidine amino acid to histamine through microbial ( morganellamorganii ) or enzymatic) proteolyticenzyme) processes under temperature ubused conditions. the aims of my termpaper to determination of histamine in fish especially tuna fish ( highest typeconsumed ) in several advanced techniques ( High pressure liquid chromatographyHPLC , thin layer chromatography TLC , gas chromatography GC , capillaryelectrophoresis CE ) and compartion these techniques with each other.Body Standard amine solution prepared byused stock solution containing different amino acid (1000mgl) diluted in HCL (0.1M)or TCA (5% trichloroacetic acid), added to it 1.
7- diaminoheptane as internalstandard to know the concentration of analyte in the standard.Sample pretreatment in capillaryelectrophoresis done by extraction and purification that’s done by homogenizedtuna fish sample with extraction solution either in HCL or in TCA then storedthe sample (at 4c for 7 days) to crystallize most of the fat, centrifugationand filtration and purification by LLE (liquid- liquid extraction) by N-butanol then the sample ready to analysis.the same thing done in HPLCbut after filtration a neceeary stepdone which is neutralization ( ph 6-7) to keep native form for amino acid thenpurification done by both ( LLE , SFE solid face extraction ) SFE done byactivation ( methanol + distilled water + HCL+ sample extract ) then washing bypotassium citrate buffer and finally elution with potassium citrate buffer –isopropanol .
before the sample injection the derivatization of pre-column by OPA ( o-phathaldialdehyde ) whichis the most common one of derivatization because it react with histamine in 15second that is very necessary to facilate detection and reduce time of analysis.separation of analyte in CE done byused capillary electrophoresis column that contain fused silica , the capillaryconducted with sodium hydroxide , water and buffer ( to make histamine morestable under acidic condition ph = 2.44) the flushed of buffer done in sequenceinterval , the capillary inlet must be closed after each flushed interval doneto reduce the time of exposed on the capillary , injection of the sample doneunder pressure , temperature constant 25c , and the voltage was graduallyramped up in different amount to minimize the possible sample loss inside thecapillary .Detection of analyte in CE happenedby diode array detector (DAD) 2mg/kg while detection in HPLC happened by bothfluorescence detector 0.5mg/kg and DAD 1mg/kg. the time used for separation of analyte in CE 9 minwhile in HPLC 20 min. the retention time used for elution histamine in CE 4minwhile in HPLC 8 min. the linearity that done by calibration curve for CE is upto 1000 mg/kg while in HPLC up to 100mg/kg .
the correlation between HPLC and CE is very good ( the correlation ofhistamine = 0.994). See (table 3). Parameter CE HPLC Sample pretreatment Extraction + purification Extraction + neutralization + purification Pre-column derivatization No Yes Correlation with HPLC 0.994 for histamine – Analysis time ( including sample pretreatment ) 15min ( 35min) 30min (90min ) Separation time 9min 20min Lower detection limit 2-6 mg /kg 1-8.5 mg /kg Liner range Up to 1000 Up to 100 analytes Histamine , tyramine Histamine, tyramine, cadaverine, putrescine, speridine, agmatine. Table 3: comparation HPLC versus CE.Preparation of standard amine doneby stock solution prepared by diluted about (0.
2-0.25g) of each amino acid in10 ml of (TCA) trichloroacetic acid.derivatizataion of standard aminedone by take 1ml of standard amine in 5ml screw cap tube added to it 1ml ofphosphate buffer (ph=9) , drop of NaOH and 2 ml of dansly reagent ( 50mg ofdansyl chloride and 10 ml acetone ).then cover test tube with aluminum ,incubated it at 55c for 1hour , to done perfect densalyation that necessary to reduced total run time .samplepretreatment in thin layer chromatography (TLC) done by extraction andderivatization , extraction happened by homogenized 10g of tuna fish sample with 30 ml of 5% hot TCA (80-90c)for 2 Min then centrifugation ,filtration the supernatant solution, 1ml of filtrate derivatization similar tostandard amine derivatization. separation of analyte (mixture of amino acid ) in TLC done by take10 microlitiers of it and applied on precoated silica gel plate (stationary phase) then added to it mobile phase (Chloroform: triethylamine (100:25)) afterthat the plate sprayed with isopropanol: triethylamine (8:2) to enhance theflouoresence.
While in HPLC the separation of analyte done by injection(5-8microlitter) of it in c18 column and added mobile phase (methanol: water(70:30)) after that to make good separation of all amine by increasing methanolconcentration gradually (75%, 80%, 100%). Detection in TLC done by UV at 365nm, while inHPLC done at 245. the lower detection limit in HPLC (5-10 ng)while in TLC (15-20 ng ) , the analysis time used for onesample in HPLC 30 min while in TLC 10 min , the number of sample that can beanalysis at same time in TLC is 10-12 comparing to HPLC one sample can analysis, so the cost in TLC lower than HPLC because the amount of reagent used lowerthan HPLC ( 1ml for one sample in TLCcomparing to HPLC 30 ml for one sample ) , also the cost of HPLC higher it needexpensive solvents, high operationalskill and careful maintenance.
the linearity done by HPLC up to 100ng while inTLC up to 300ng , the repapilty for HPLC lower than 3% while in TLC lower than8% ,the senseviety of HPLC 9.35 while TLC 0.84 , the correlation for HPLC ( 0.998-0.999) while TLC (0.
996-0.999) that’smean there are marginal differences between two method. (See table 4) . parameter HPLC TLC linearity 5-100ng 20-300ng senseviety 9.35 0.84 repapilty Lee than 3% less than 8% time used for analysis 30 min 10min coast expensive inexpensive number of sample that can analysis at same time 1 10-12 in one plate lower detection limit 5-10ng 15-20ng amount of reagent used for analysis one sample 30ml 4ml correlation coefficient (0.
998-0.999) (0.996-0.999) (Table 4: compartion HPLC versus TLC).Preparation of standard solution ingas chromatography done by dissolving 50mg of internal standard (1, 9 nanodiol)and histamine in 100 ml methanol. compared these with CE they useddifferent internal standard which is(1.7-diaminoheptane ) while in GC ( 1,9 nanodiol ) that is necessary for bothtechniques to known concentration of analyte in the standard , while in HPLCstandard solution can prepared with and without internal standard accordingto the aims of the analysis , while inthin layer chromatography they don’t used internal standard because the systemof it is very simply compared to other techniques it does not contain columnonly stationary phase formed as plate . sample pretreatment in GC done by extractionand derivatization that’s done by dissolving sample of tuna fish in alkalinemethanol after that the extractor sample derivatization with pentafluoropionicanhydride to make it volatile , compared to HPLC derivatization of pre-columnnecessary to rapid analysis procedure , also in TLC derivatization is verynecessary to reduce time of analysis while in CE derivatization is notnecessary and not applied because during analysis buffer flushed to makehistamine more stable and to reduce amount of lost histamine .
Injection of sample done by usedthree different polarity columns (Cp-sill 5CB, Cp-sill 8CB, and CP-sill 9CB)and nitrogen gas used to make separation and to make reactant betweenstationary phase and analyte more active under oven condition (160-280c) andthe separation done according to polarity.Detection done by flame ionization detector(FID) the lower detection limit (5microgram /g). the analysis time used toanalysis less than 20 min compared toanother technique ( HPLC 30min ,TLC10min, CE 15 min ) which is the third one arrangement according to consuming time , the retention time used todetect histamine differ according to column used ( 4.66min, 4.77min,5.24min ) asthe above arrangement used , accordingto that CP-sil5cB the more efficient column used to detection histamine comparingto another columns , recovery for it ( 2.7-7.
8% ) . (See table 5). Parameter CE HPLC TLC GC Sample pretreatment Extraction , purification Extraction, neutralization, purification, derivatization.
Extraction, derivatization. Extraction, derivatization. Internal standard Yes Can do with it and with out of it No Yes Derivatization used No Yes OPA (o-phathaldialdehyde ) Yes (50 mg of dansyl chloride and 10 ml acetone). Yes pentafluoropionic anhydride Column yes Yes no Yes Stationary phase Silica C18 Silica (Cp-sill 5CB, Cp-sill 8CB, CP-sill 9CB). Mobile phase Liquid (buffer , NaOH , water ) Liquid ( water + methanol ) Liquid (chloroform: triethylamine) Inert gas (nitrogen gas ) Analysis time 15 min 30 min 10 min 20 min Lower detection limit 2-6 mg /kg 5-10 ng 15-20 ng 5 microgram /g Detector diode array detector ( DAD) Fluorescence detector and diode array detector. by UV flame ionization detector(FID) Linearity Up to 1000 ng Up to 100 ng Up to 300ng — Reagent used Low High Low low Table 5: CE, TLC, HPLC, GCcomparison.
the most problem that’s happened inHPLC method is derivatization step that’s need more time to done which isrequired controlled reaction conditions , also can effect peak brooding and sample dilution , so analysis withoutderivatization is not improved good detection . HPLC method need high technicalskill, time and cost, it’s more applicable in determine information about thecomposition of biogenic amine in food sample, that’s mean it’s suitable forfood quality control.TLC method that’s features in rapidmethod which required 10 min , and less expensive so it’s sutabile for analysisdifferent sample at the same time , according to that it’s more applicable infish industry to detect biogenic amine especially toxic histamine. GC method that’s accurate method, quicker thanHPLC about 20 min need to complete analysis, it’s more applicable in determinehistamine and another biogenic amine in fish and other food.CE has higher theoreticalresolution than other analysis methods , no organic solvent used so the cost ofreagent and capplieries is reduced , quicker than HPLC and GC , more accuratemethod no interference without result , not required derivatization, its applicable to determine histamineconcentration in marine product .Conclusion According to previous, histamineproduced in food by decarboxylation of histidine amino acid to histaminethrough microbial or enzymatic process under temperature ubused conditions. histamine can determine by severalmethods (HPLC, CE, TLC, GC) according to the comparison between these methodthe best one is CE that’s the highest theoretical resolution than other method,no organic solvent used so coast reduced, quicker than HPLC and GC time consumed15min, more accurate method no interference between the result, not requiredderivatization step that’s consumed more time.